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Catalano A, Haas LS, Zodel K, Adlesic M, Cuomo F, Peighambari A, Metzger P, Huang H, Haug S, Köttgen A, Köhler N, Boerries M, Frew IJ. Mutations in tumor suppressor genes Vhl and Rassf1a cause DNA damage, chromosomal instability and induce gene expression changes characteristic of clear cell renal cell carcinoma. Kidney Int 2025; 107:666-686. [PMID: 39725222 DOI: 10.1016/j.kint.2024.12.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Revised: 11/06/2024] [Accepted: 12/04/2024] [Indexed: 12/28/2024]
Abstract
RASSF1A is frequently biallelically inactivated in clear cell renal cell carcinoma (ccRCC) due to loss of chromosome 3p and promoter hypermethylation. Here we investigated the cellular and molecular consequences of single and combined deletion of the Rassf1a and Vhl tumor suppressor genes to model the common ccRCC genotype of combined loss of function of RASSF1A and VHL. In mouse embryonic fibroblasts and in primary kidney epithelial cells, double deletion of Rassf1a and Vhl caused chromosomal segregation defects and increased formation of micronuclei, demonstrating that pVHL and RASSF1A function to maintain genomic integrity. Combined Rassf1a and Vhl deletion in kidney epithelial cells in vivo increased proliferation and caused mild tubular disorganization, but did not lead to the development of kidney tumors. Single cell RNA-sequencing unexpectedly revealed that Rassf1a or Vhl deletion both induce the expression of an overlapping set of genes in a sub-population of proximal tubule cells. Many of these genes are also upregulated in the Vhl/Trp53/Rb1 deficient mouse model of ccRCC. In other subsets of proximal tubule cells, combined Vhl/Rassf1a deletion induced the expression of additional genes that were not upregulated in each of the single knockouts. The expression of the human homologues of Rassf1a-regulated genes correlate negatively with RASSF1 expression levels in human ccRCC. Our results suggest that the loss of RASSF1A function establishes a ccRCC-characteristic gene expression pattern.
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Affiliation(s)
- Antonella Catalano
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Laura S Haas
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Kyra Zodel
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Mojca Adlesic
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Francesca Cuomo
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Asin Peighambari
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Patrick Metzger
- Institute of Medical Bioinformatics and Systems Medicine, Medical Centre-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Hsin Huang
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Stefan Haug
- Institute of Genetic Epidemiology, Faculty of Medicine and Medical Center-University of Freiburg, Freiburg, Germany
| | - Anna Köttgen
- Institute of Genetic Epidemiology, Faculty of Medicine and Medical Center-University of Freiburg, Freiburg, Germany
| | - Natalie Köhler
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Melanie Boerries
- Institute of Medical Bioinformatics and Systems Medicine, Medical Centre-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; Comprehensive Cancer Center Freiburg (CCCF), Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK), Partner Site Freiburg, Freiburg, Germany
| | - Ian J Frew
- Clinic of Internal Medicine I, Hematology, Oncology and Stem Cell Transplantation, Medical Centre - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; Comprehensive Cancer Center Freiburg (CCCF), Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK), Partner Site Freiburg, Freiburg, Germany; Signalling Research Centres BIOSS and CIBSS, Faculty of Biology University of Freiburg, Freiburg, Germany.
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Li Z, Yin B, Xu Y, Wang C, Li X, Lu S, Ke S, Qian B, Yu H, Bai M, Li Z, Zhou Y, Jiang H, Ma Y. Von Hippel-Lindau deficiency protects the liver against ischemia/reperfusion injury through the regulation of hypoxia-inducible factor 1α and 2α. Hepatol Commun 2024; 8:e0567. [PMID: 39585306 PMCID: PMC11596652 DOI: 10.1097/hc9.0000000000000567] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Accepted: 08/25/2024] [Indexed: 11/26/2024] Open
Abstract
BACKGROUND Ischemia and reperfusion (I/R)-induced liver injury contributes to morbidity and mortality during hepatic surgery or liver transplantation. As a pivotal regulator of cancer and inflammation, the role of Von Hippel-Lindau (VHL) in hepatic I/R injury remains undetermined. METHODS We investigated the role of VHL in hepatic I/R injury by generating VHL conditional knockout (VHL-KO) mice. The downstream mechanisms of VHL were confirmed, and the role of HIF-2α in hepatic I/R injury was further investigated. RESULTS In this study, we discovered that VHL upregulation was associated with hepatic I/R injury in a mouse model. VHL gene knockout (VHL-KO) and overexpression (Ad-VHL) mice demonstrated that VHL aggravated liver injury, increased inflammation, and accelerated cell death in hepatic I/R injury. The VHL protein (pVHL) regulates a crucial control mechanism by targeting HIFα subunits for ubiquitin-mediated degradation. In vitro and in vivo studies demonstrated that VHL interacted with and repressed hypoxia-inducible factor 1α (HIF-1α) and hypoxia-inducible factor 2α (HIF-2α) expression during hepatic I/R injury. Notably, the inhibition of HIF-1α or 2α, as well as the concurrent inhibition of HIF-1α and 2α, abrogated the protective effect of VHL-KO. The severe stabilization of HIF-1α or 2α, as well as the simultaneous overexpression of HIF-1α and 2α, compensated for the detrimental effect of VHL. CONCLUSIONS Thus, we identified the VHL-HIF-1α/HIF-2α axis as an indispensable pathway that may be a novel target for mediating hepatic I/R injury.
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Affiliation(s)
- Zihao Li
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Bing Yin
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Yanan Xu
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Hepatopancreatobiliary Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Chaoqun Wang
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Army Medical University, Chongqing, China
| | - Xinglong Li
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Shounan Lu
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Shanjia Ke
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Department of Hepatobiliary Surgery, Peking University People’s Hospital, Beijing, China
| | - Baolin Qian
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Hongjun Yu
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Miaoyu Bai
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Zhongyu Li
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Yongzhi Zhou
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Hongchi Jiang
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Yong Ma
- Department of Minimally Invasive Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, The First Affiliated Hospital of Harbin Medical University, Harbin, China
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Kurlekar S, Lima JDCC, Li R, Lombardi O, Masson N, Barros AB, Pontecorvi V, Mole DR, Pugh CW, Adam J, Ratcliffe PJ. Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type-Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney. Cancer Res 2024; 84:1799-1816. [PMID: 38502859 PMCID: PMC11148546 DOI: 10.1158/0008-5472.can-23-3248] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2023] [Revised: 01/16/2024] [Accepted: 03/13/2024] [Indexed: 03/21/2024]
Abstract
Defining the initial events in oncogenesis and the cellular responses they entrain, even in advance of morphologic abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this, we longitudinally studied the changes induced by loss of the tumor suppressor gene von Hippel Lindau (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl inactivation was directly coupled to expression of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell type-specific responses to Vhl inactivation, defined a proximal tubular cell class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell tagging also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Overall, this study reports an experimental strategy for understanding oncogenic processes in which cells bearing genetic alterations can be generated in their native context, marked, and analyzed over time. The observed effects of loss of Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma. SIGNIFICANCE Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.
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Affiliation(s)
- Samvid Kurlekar
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Joanna D C C Lima
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Ran Li
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Olivia Lombardi
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Norma Masson
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Ayslan B Barros
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Virginia Pontecorvi
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - David R Mole
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Christopher W Pugh
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Julie Adam
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Peter J Ratcliffe
- Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
- The Francis Crick Institute, 1 Midland Road, London, United Kingdom
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HIF-1α and HIF-2α differently regulate tumour development and inflammation of clear cell renal cell carcinoma in mice. Nat Commun 2020; 11:4111. [PMID: 32807776 PMCID: PMC7431415 DOI: 10.1038/s41467-020-17873-3] [Citation(s) in RCA: 173] [Impact Index Per Article: 34.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Accepted: 07/21/2020] [Indexed: 02/06/2023] Open
Abstract
Mutational inactivation of VHL is the earliest genetic event in the majority of clear cell renal cell carcinomas (ccRCC), leading to accumulation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCC and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Herein we show using an autochthonous ccRCC model that Hif1a is essential for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are required for the clear cell phenotype. Transcriptomic and proteomic analyses reveal that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. HIF-2α-deficient tumours are characterised by increased antigen presentation, interferon signalling and CD8+ T cell infiltration and activation. Single copy loss of HIF1A or high levels of HIF2A mRNA expression correlate with altered immune microenvironments in human ccRCC. These studies reveal an oncogenic role of HIF-1α in ccRCC initiation and suggest that alterations in the balance of HIF-1α and HIF-2α activities can affect different aspects of ccRCC biology and disease aggressiveness.
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Gläsker S, Vergauwen E, Koch CA, Kutikov A, Vortmeyer AO. Von Hippel-Lindau Disease: Current Challenges and Future Prospects. Onco Targets Ther 2020; 13:5669-5690. [PMID: 32606780 PMCID: PMC7305855 DOI: 10.2147/ott.s190753] [Citation(s) in RCA: 56] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2020] [Accepted: 05/20/2020] [Indexed: 12/13/2022] Open
Abstract
Understanding of molecular mechanisms of tumor growth has an increasing impact on the development of diagnostics and targeted therapy of human neoplasia. In this review, we summarize the current knowledge on molecular mechanisms and their clinical implications in von Hippel-Lindau (VHL) disease. This autosomal dominant tumor syndrome usually manifests in young adulthood and predisposes affected patients to the development of benign and malignant tumors of different organ systems mainly including the nervous system and internal organs. A consequent screening and timely preventive treatment of lesions are crucial for patients affected by VHL disease. Surgical indications and treatment have been evaluated and optimized over many years. In the last decade, pharmacological therapies have been evolving, but are largely still at an experimental stage. Effective pharmacological therapy as well as detection of biomarkers is based on the understanding of the molecular basis of disease. The molecular basis of von Hippel-Lindau disease is the loss of function of the VHL protein and subsequent accumulation of hypoxia-inducible factor with downstream effects on cellular metabolism and differentiation. Organs affected by VHL disease may develop frank tumors. More characteristically, however, they reveal multiple separate microscopic foci of neoplastic cell proliferation. The exact mechanisms of tumorigenesis in VHL disease are, however, still not entirely understood and knowledge on biomarkers and targeted therapy is scarce.
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Affiliation(s)
- Sven Gläsker
- Neurosurgical Practise Lake Constance, Singen (Hohentwiel), Germany.,Department of Neurosurgery, VUB University Medical Center Brussels, Brussels, Belgium
| | - Evelynn Vergauwen
- Department of Neurosurgery, VUB University Medical Center Brussels, Brussels, Belgium.,Department of Neurology, University Hospital Antwerp, Antwerp, Belgium
| | | | | | - Alexander O Vortmeyer
- Department of Pathology, Indiana University-Purdue University, Indianapolis, IN, USA
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Neddylation inhibitor MLN4924 suppresses cilia formation by modulating AKT1. Protein Cell 2019; 10:726-744. [PMID: 30850948 PMCID: PMC6776484 DOI: 10.1007/s13238-019-0614-3] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2018] [Accepted: 01/24/2019] [Indexed: 11/25/2022] Open
Abstract
The primary cilium is a microtubule-based sensory organelle. The molecular mechanism that regulates ciliary dynamics remains elusive. Here, we report an unexpected finding that MLN4924, a small molecule inhibitor of NEDD8-activating enzyme (NAE), blocks primary ciliary formation by inhibiting synthesis/assembly and promoting disassembly. This is mainly mediated by MLN4924-induced phosphorylation of AKT1 at Ser473 under serum-starved, ciliary-promoting conditions. Indeed, pharmaceutical inhibition (by MK2206) or genetic depletion (via siRNA) of AKT1 rescues MLN4924 effect, indicating its causal role. Interestingly, pAKT1-Ser473 activity regulates both ciliary synthesis/assembly and disassembly in a MLN4924 dependent manner, whereas pAKT-Thr308 determines the ciliary length in MLN4924-independent but VHL-dependent manner. Finally, MLN4924 inhibits mouse hair regrowth, a process requires ciliogenesis. Collectively, our study demonstrates an unexpected role of a neddylation inhibitor in regulation of ciliogenesis via AKT1, and provides a proof-of-concept for potential utility of MLN4924 in the treatment of human diseases associated with abnormal ciliogenesis.
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Maniaci C, Hughes SJ, Testa A, Chen W, Lamont DJ, Rocha S, Alessi DR, Romeo R, Ciulli A. Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat Commun 2017; 8:830. [PMID: 29018234 PMCID: PMC5635026 DOI: 10.1038/s41467-017-00954-1] [Citation(s) in RCA: 180] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2017] [Accepted: 08/01/2017] [Indexed: 01/09/2023] Open
Abstract
E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for ‘proteolysis-targeting chimeras’). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases. Targeting the ubiquitin proteasome system to modulate protein homeostasis using small molecules has promising therapeutic potential. Here the authors describe Homo-PROTACS: small molecules that can induce the homo-dimerization of E3 ubiquitin ligases and cause their proteasome-dependent degradation.
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Affiliation(s)
- Chiara Maniaci
- Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK.,Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK
| | - Scott J Hughes
- Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK
| | - Andrea Testa
- Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK
| | - Wenzhang Chen
- Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK
| | - Douglas J Lamont
- Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK
| | - Sonia Rocha
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, DD1 5EH, Scotland, UK
| | - Dario R Alessi
- Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK
| | - Roberto Romeo
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, Polo Universitario Viale SS. Annunziata SNC, Messina, 98168, Italy
| | - Alessio Ciulli
- Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH, UK.
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Zhou Q, Chen T, Zhang W, Bozkanat M, Li Y, Xiao L, van Breemen RB, Christman JW, Sznajder JI, Zhou G. Suppression of von Hippel-Lindau Protein in Fibroblasts Protects against Bleomycin-Induced Pulmonary Fibrosis. Am J Respir Cell Mol Biol 2016; 54:728-39. [PMID: 26488390 PMCID: PMC4942192 DOI: 10.1165/rcmb.2015-0111oc] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Accepted: 10/16/2015] [Indexed: 12/20/2022] Open
Abstract
We have reported that von Hippel-Lindau protein (pVHL) expression is elevated in human and mouse fibrotic lungs and that overexpression of pVHL stimulates fibroblast proliferation. We sought to determine whether loss of pVHL in fibroblasts prevents injury and fibrosis in mice that are treated with bleomycin. We generated heterozygous fibroblast-specific pVHL (Fsp-VHL) knockdown mice (Fsp-VHL(+/-)) and homozygous Fsp-VHL knockout mice (Fsp-VHL(-/-)) by crossbreeding vhlh 2-lox mice (VHL(fl/fl)) with Fsp-Cre recombinase mice. Our data show that Fsp-VHL(-/-) mice, but not Fsp-VHL(+/-) mice, have elevated red blood cell counts, hematocrit, hemoglobin content, and expression of hypoxia-inducible factor (HIF) targets, indicating HIF activation. To examine the role of pVHL in bleomycin-induced lung injury and fibrosis in vivo, we administered PBS or bleomycin to age-, sex-, and strain-matched 8-week-old VHL(fl/fl), Fsp-VHL(+/-), and Fsp-VHL(-/-) mice. In Fsp-VHL(+/-) and Fsp-VHL(-/-) mice, bleomycin-induced collagen accumulation, fibroblast proliferation, differentiation, and matrix protein dysregulation were markedly attenuated. Suppression of pVHL also decreased bleomycin-induced Wnt signaling and prostaglandin E2 signaling but did not affect bleomycin-induced initial acute lung injury and lung inflammation. These results indicate that pVHL has a pivotal role in bleomycin-induced pulmonary fibrosis, possibly via an HIF-independent pathway. Paradoxically, pVHL does not affect bleomycin-induced lung injury and inflammation, indicating a separation of the mechanisms involved in injury/inflammation from those involved in pulmonary fibrosis.
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Affiliation(s)
| | | | - Wei Zhang
- Department of Preventive Medicine and
| | | | | | - Lei Xiao
- Department of Medicine, Division of Pulmonary, Critical Care, Sleep, and Allergy, and
| | | | - John W. Christman
- Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Ohio State University, Columbus, Ohio
| | - Jacob I. Sznajder
- Department of Medicine, Division of Pulmonary and Critical Care Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois; and
| | - Guofei Zhou
- Departments of Pediatrics and
- Cancer Center, University of Illinois at Chicago, Chicago, Illinois
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Jiang X, Zhang D, Zhang H, Huang Y, Teng M. Role of Ran-regulated nuclear-cytoplasmic trafficking of pVHL in the regulation of microtubular stability-mediated HIF-1α in hypoxic cardiomyocytes. Sci Rep 2015; 5:9193. [PMID: 25779090 PMCID: PMC4361876 DOI: 10.1038/srep09193] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Accepted: 02/25/2015] [Indexed: 12/30/2022] Open
Abstract
Our previous study suggested that microtubule network alteration affects the process of glycolysis in cardiomyocytes (CMs) via the regulation of hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known regarding the underlying mechanisms of microtubule network alteration-induced changes of HIF-1α. The von Hippel–Lindau tumor suppressor protein (pVHL) has been shown to mediate the ubiquitination of HIF-1α in the nuclear compartment prior to HIF-1α exportation to the cytoplasm, and pVHL dynamic nuclear-cytoplasmic trafficking is indicated to be involved in the process of HIF-1α degradation. In this study, by administering different microtubule-stabilizing and -depolymerizing interventions, we demonstrated that microtubule stabilization promoted pVHL nuclear export and drove the translocation of pVHL to the cytoplasm, while microtubule disruption prevented pVHL nuclear export in hypoxic CMs. Moreover, the ratio between nuclear and cytoplasmic pVHL was associated with HIF-1α regulation. Importantly, microtubule network alteration also affected the subcellular localization of Ran, which was involved in the regulation of pVHL nuclear-cytoplasmic trafficking. The above results suggest that the subcellular translocation of pVHL plays an important role in microtubular structure alteration-induced HIF-1α regulation. Interestingly, Ran is involved in the process of pVHL nuclear-cytoplasmic trafficking following microtubule network alteration in hypoxic CMs.
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Affiliation(s)
- Xupin Jiang
- 1] Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, The Third Military Medical University, Chongqing, China [2] Department of Burn and Plastic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Dongxia Zhang
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, The Third Military Medical University, Chongqing, China
| | - Hengshu Zhang
- Department of Burn and Plastic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Yuesheng Huang
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, The Third Military Medical University, Chongqing, China
| | - Miao Teng
- Department of Burn and Plastic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
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10
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Ding XF, Zhou J, Hu QY, Liu SC, Chen G. The tumor suppressor pVHL down-regulates never-in-mitosis A-related kinase 8 via hypoxia-inducible factors to maintain cilia in human renal cancer cells. J Biol Chem 2014; 290:1389-94. [PMID: 25451921 DOI: 10.1074/jbc.m114.589226] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.
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Affiliation(s)
| | - Jun Zhou
- From the School of Medicine, the Institute of Tumor, and
| | | | - Shuang-Chun Liu
- the Taizhou Municipal Hospital, Taizhou, Zhejiang, 318000 China
| | - Guang Chen
- From the School of Medicine, the Institute of Tumor, and the School of Pharmaceutical and Chemical Engineering, Taizhou University, Taizhou, Zhejiang 318000 and
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11
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Schmid S, Gillessen S, Binet I, Brändle M, Engeler D, Greiner J, Hader C, Heinimann K, Kloos P, Krek W, Krull I, Stoeckli SJ, Sulz MC, van Leyen K, Weber J, Rothermundt C, Hundsberger T. Management of von hippel-lindau disease: an interdisciplinary review. Oncol Res Treat 2014; 37:761-71. [PMID: 25531723 DOI: 10.1159/000369362] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2014] [Accepted: 10/22/2014] [Indexed: 12/11/2022]
Abstract
Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited tumour predisposition syndrome with an incidence of 1:36,000 newborns, the estimated prevalence in Europe is about 1-9/100,000. It is associated with an increased risk of developing various benign and malignant tumours, thus affecting multiple organs at different time points in the life of a patient. Disease severity and diversity as well as age at first symptoms vary considerably, and diagnostic delay due to failure of recognition is a relevant issue. The identification of a disease-causing VHL germline mutation subsequently allows family members at risk to undergo predictive genetic testing after genetic counselling. Clinical management of patients and families should optimally be offered as an interdisciplinary approach. Prophylactic screening programs are a cornerstone of care, and have markedly improved median overall survival of affected patients. The aim of this review is to give an overview of the heterogeneous manifestations of the VHL syndrome and to highlight the diagnostic and therapeutic challenges characteristic for this orphan disease. A comprehensive update of the underlying genetic and molecular principles is additionally provided. We also describe how the St. Gallen VHL multidisciplinary group is organised as an example of interdisciplinary cooperation in a tertiary hospital in Switzerland.
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Affiliation(s)
- Sabine Schmid
- Division of Haematology and Oncology, Cantonal Hospital St. Gallen, Switzerland
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12
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Interaction between Nm23 and the tumor suppressor VHL. Naunyn Schmiedebergs Arch Pharmacol 2014; 388:143-52. [PMID: 24915993 DOI: 10.1007/s00210-014-1002-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2014] [Accepted: 06/02/2014] [Indexed: 01/01/2023]
Abstract
Among the anti-tumor genes (tumor suppressors and metastasis suppressors), the von-Hippel Lindau gene and the Nm23 family of genes are among the more intriguing ones. Both are small (long and short forms of VHL are 30 and 19 kD, respectively, and Nm23 is ~17 kD), and both possess diverse molecular and cellular functions. Despite extensive studies, the entire spectra of functions and the molecular function-phenotype correlation of these two proteins have not been completely elucidated. In this report, we present data showing these two proteins interact physically. We also summarize and confirm the previous studies that demonstrated the endocytic function of these two genes and further show that the endocytic function of VHL is mediated through the activity of Nm23. These functional and molecular interactions are evolutionarily conserved from Drosophila to human.
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13
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Minervini G, Panizzoni E, Giollo M, Masiero A, Ferrari C, Tosatto SCE. Design and analysis of a Petri net model of the Von Hippel-Lindau (VHL) tumor suppressor interaction network. PLoS One 2014; 9:e96986. [PMID: 24886840 PMCID: PMC4041725 DOI: 10.1371/journal.pone.0096986] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2013] [Accepted: 04/14/2014] [Indexed: 02/01/2023] Open
Abstract
Von Hippel-Lindau (VHL) syndrome is a hereditary condition predisposing to the development of different cancer forms, related to germline inactivation of the homonymous tumor suppressor pVHL. The best characterized function of pVHL is the ubiquitination dependent degradation of Hypoxia Inducible Factor (HIF) via the proteasome. It is also involved in several cellular pathways acting as a molecular hub and interacting with more than 200 different proteins. Molecular details of pVHL plasticity remain in large part unknown. Here, we present a novel manually curated Petri Net (PN) model of the main pVHL functional pathways. The model was built using functional information derived from the literature. It includes all major pVHL functions and is able to credibly reproduce VHL syndrome at the molecular level. The reliability of the PN model also allowed in silico knockout experiments, driven by previous model analysis. Interestingly, PN analysis suggests that the variability of different VHL manifestations is correlated with the concomitant inactivation of different metabolic pathways.
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Affiliation(s)
| | | | - Manuel Giollo
- Dept. of Biomedical Sciences, University of Padua, Padua, Italy
- Dept. of Information Engineering, University of Padua, Padua, Italy
| | | | - Carlo Ferrari
- Dept. of Information Engineering, University of Padua, Padua, Italy
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14
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Jung YS, Lee SJ, Lee SH, Chung JY, Jung YJ, Hwang SH, Ha NC, Park BJ. Loss of VHL promotes progerin expression, leading to impaired p14/ARF function and suppression of p53 activity. Cell Cycle 2014; 12:2277-90. [PMID: 24067370 DOI: 10.4161/cc.25371] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Renal cell carcinomas (RCCs) are frequently occurring genitourinary malignancies in the aged population. A morphological characteristic of RCCs is an irregular nuclear shape, which is used to index cancer grades. Other features of RCCs include the genetic inactivation of the von Hippel-Lindau gene, VHL, and p53 genetic-independent inactivation. An aberrant nuclear shape or p53 suppression has not yet been demonstrated. We examined the effect of progerin (an altered splicing product of the LMNA gene linked to Hutchinson Gilford progeria syndrome; HGPS) on the nuclear deformation of RCCs in comparison to that of HGPS cells. In this study, we showed that progerin was suppressed by pVHL and was responsible for nuclear irregularities as well as p53 inactivation. Thus, progerin suppression can ameliorate nuclear abnormalities and reactivate p53 in response to genotoxic addition. Furthermore, we found that progerin was a target of pVHL E3 ligase and suppressed p53 activity by p14/ARF inhibition. Our findings indicate that the elevated expression of progerin in RCCs results from the loss of pVHL and leads to p53 inactivation through p14/ARF suppression. Interestingly, we showed that progerin was expressed in human leukemia and primary cell lines, raising the possibility that the expression of this LMNA variant may be a common event in age-related cancer progression.
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Affiliation(s)
- Youn-Sang Jung
- Department of Molecular Biology; College of Natural Science, Pusan National University; Busan, Republic of Korea
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15
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Abstract
pVHL is a downstream target of E2F1, which harbours an E2F1-binding site in its promoter. Moreover, pVHL binds to E2F1 in vitro and in vivo, resulting in inhibition of E2F1 transcriptional activity.
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16
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Chen T, Sun M, Zhou G. Von Hippel-Lindau protein and respiratory diseases. World J Respirol 2013; 3:48-56. [DOI: 10.5320/wjr.v3.i3.48] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/27/2013] [Revised: 07/09/2013] [Accepted: 07/19/2013] [Indexed: 02/06/2023] Open
Abstract
Von Hippel-Lindau protein (pVHL) was first identified as a tumor suppressor gene as mutations in the VHL gene predispose individuals to systemic benign or malignant tumors and cysts in many organs, including renal cell carcinoma of the clear-cell type and hemangioblastoma. Although pVHL is best known to act as a component of ubiquitin protein ligase for the proteasomal degradation of hypoxia inducible factor (HIF)-α, pVHL also interacts with extracellular matrix proteins and cytoskeleton, regulating extracellular matrix assembly, cell signaling, and many other cellular functions. Recent studies suggest that pVHL contributes to many lung diseases, including pulmonary arterial hypertension, lung cancer, pulmonary fibrosis, and acute respiratory distress syndrome. Mutation or loss of function of pVHL activates HIF and induced expression of vascular endothelial growth factor, endothelin-1, and FoxM1, leading to pulmonary arterial hypertension. Loss of pVHL in lung cancer cells promotes epithelial-mesenchymal transition and cancer migration and invasion while decreasing lung cancer cell proliferation and colonization. In patients of idiopathic pulmonary fibrosis, elevated expression of pVHL induces expression of fibronectin/integrin α5β1/focal adhesion kinase signaling, resulting in fibroproliferation and fibrosis. In alveolar epithelial cells, pVHL mediates Na-K-ATPase degradation in an HIF independent pathway, causing decreased edema clearance during hypoxia. These studies suggest that pVHL plays key roles in the pathogenesis of many lung diseases, and further investigations are warranted to elucidate the underlying molecular mechanisms.
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17
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Genomics and epigenomics of clear cell renal cell carcinoma: recent developments and potential applications. Cancer Lett 2013; 341:111-26. [PMID: 23933176 DOI: 10.1016/j.canlet.2013.08.006] [Citation(s) in RCA: 92] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2013] [Revised: 07/12/2013] [Accepted: 08/02/2013] [Indexed: 12/21/2022]
Abstract
Majority of clear cell renal cell carcinomas (ccRCCs) are diagnosed in the advanced metastatic stage resulting in dramatic decrease of patient survival. Thereby, early detection and monitoring of the disease may improve prognosis and treatment results. Recent technological advances enable the identification of genetic events associated with ccRCC and reveal significant molecular heterogeneity of ccRCC tumors. This review summarizes recent findings in ccRCC genomics and epigenomics derived from chromosomal aberrations, DNA sequencing and methylation, mRNA, miRNA expression profiling experiments. We provide a molecular insight into ccRCC pathology and recapitulate possible clinical applications of genomic alterations as predictive and prognostic biomarkers.
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18
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Marques I, Teixeira AL, Ferreira M, Assis J, Lobo F, Maurício J, Medeiros R. Influence of survivin (BIRC5) and caspase-9 (CASP9) functional polymorphisms in renal cell carcinoma development: a study in a southern European population. Mol Biol Rep 2013; 40:4819-26. [PMID: 23645041 DOI: 10.1007/s11033-013-2578-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2012] [Accepted: 04/29/2013] [Indexed: 01/08/2023]
Abstract
Renal cell carcinoma (RCC) is the most common cancer of the adult kidney and its incidence and mortality has increase in the last 20 years. The disruption of cellular death is one the mechanism involved in cancer development. This process is precise regulated by apoptotic and anti-apoptotic molecules. Survivin (BIRC5) is a member of the inhibitor of apoptosis protein family and has the ability to inhibit the activation of the pro-apoptotic caspase-9 (CASP9). Thus BIRC5 and CASP9 functional polymorphisms might modulate the apoptosis and consequently RCC development. Our purpose was to investigate the potential role of BIRC5-31G/C and CASP9+83C/T functional polymorphisms in the risk for the development of RCC and metastatic disease. We studied the BIRC5-31G/C and CASP9+83C/T functional polymorphisms by PCR-RFLP and allelic discrimination using the 7300 real-time polymerase chain reaction system, respectively, in 178 RCC patients and in 305 healthy individuals. Regarding the BIRC5-31G/C polymorphism, there is a trend to an overrepresentation of CC genotype in RCC group compared with normal controls (aOR, 1.94; P=0.053). We observed, after gender stratification and age-adjustment, that BIRC5-31CC and CASP9+83CT/TT genotypes were associated with an increased risk for RCC development in the female group of our southern European study population (aOR=3.85; P=0.019; aOR=2.98; P=0.028; respectively). Concerning the waiting time for onset of metastatic disease, we observed that BIRC5-31CC homozygous developed metastasis 8 years earlier than the G carriers using a Cox proportional hazard model with gender as covariate (HR=4.9, P=0.038, P bootstrap=0.009). The Cox regression proportional hazard model was validated using bootstrap statistic with 1,000 samples of the same number of patients as the original dataset. Our results suggest that individual differences influence the susceptibility to RCC and tumor behavior. This genetic profile may help to define higher risk groups that would benefit from individualized chemoprevention strategies and therapies.
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Affiliation(s)
- Inês Marques
- Grupo de Oncologia Molecular-CI, Edifício Laboratórios, Instituto Português de Oncologia do Porto Francisco Gentil, EPE, 4º piso, Rua Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal
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19
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Albers J, Rajski M, Schönenberger D, Harlander S, Schraml P, von Teichman A, Georgiev S, Wild PJ, Moch H, Krek W, Frew IJ. Combined mutation of Vhl and Trp53 causes renal cysts and tumours in mice. EMBO Mol Med 2013; 5:949-64. [PMID: 23606570 PMCID: PMC3779454 DOI: 10.1002/emmm.201202231] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2012] [Revised: 03/08/2013] [Accepted: 03/12/2013] [Indexed: 12/03/2022] Open
Abstract
The combinations of genetic alterations that cooperate with von Hippel–Lindau (VHL) mutation to cause clear cell renal cell carcinoma (ccRCC) remain poorly understood. We show that the TP53 tumour suppressor gene is mutated in approximately 9% of human ccRCCs. Combined deletion of Vhl and Trp53 in primary mouse embryo fibroblasts causes proliferative dysregulation and high rates of aneuploidy. Deletion of these genes in the epithelium of the kidney induces the formation of simple cysts, atypical cysts and neoplasms, and deletion in the epithelia of the genital urinary tract leads to dysplasia and tumour formation. Kidney cysts display a reduced frequency of primary cilia and atypical cysts and neoplasms exhibit a pro-proliferative signature including activation of mTORC1 and high expression of Myc, mimicking several cellular and molecular alterations seen in human ccRCC and its precursor lesions. As the majority of ccRCC is associated with functional inactivation of VHL, our findings suggest that for a subset of ccRCC, loss of p53 function represents a critical event in tumour development.
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Affiliation(s)
- Joachim Albers
- Institute of Physiology, University of Zurich, Zurich, Switzerland
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20
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Vortmeyer AO, Falke EA, Gläsker S, Li J, Oldfield EH. Nervous system involvement in von Hippel-Lindau disease: pathology and mechanisms. Acta Neuropathol 2013; 125:333-50. [PMID: 23400300 DOI: 10.1007/s00401-013-1091-z] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2012] [Revised: 01/25/2013] [Accepted: 01/26/2013] [Indexed: 02/06/2023]
Abstract
Patients with von Hippel-Lindau disease carry a germline mutation of the Von Hippel-Lindau (VHL) tumor-suppressor gene. We discuss the molecular consequences of loss of VHL gene function and their impact on the nervous system. Dysfunction of the VHL protein causes accumulation and activation of hypoxia inducible factor (HIF) which can be demonstrated in earliest stages of tumorigenesis and is followed by expression of VEGF, erythropoietin, nitric oxide synthase and glucose transporter 1 in VHL-deficient tumor cells. HIF-independent functions of VHL, epigenetic inactivation of VHL, pVHL proteostasis, and links between loss of VHL function and developmental arrest are also described. A most intriguing feature in VHL disease is the occurrence of primary hemangioblastic tumors in the nervous system, the origin of which has not yet been entirely clarified, and current hypotheses are discussed. Endolymphatic sac tumors may extend into the brain, but originally arise from proliferation of endolymphatic duct/sac epithelium; the exact nature of the proliferating epithelial cell, however, also has remained unclear, as well as the question why tumors almost consistently develop in the intraosseous portion of the endolymphatic sac/duct only. The epitheloid clear cell morphology of both advanced hemangioblastoma and renal clear cell carcinoma can make the differential diagnosis challenging, recent developments in immunohistochemical differentiation are discussed. Finally, metastasis to brain may not only be caused by renal carcinoma, but may derive from VHL disease-associated pheochromocytoma/paraganglioma, or pancreatic neuroendocrine tumor.
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Affiliation(s)
- Alexander O Vortmeyer
- Department of Pathology, Yale University School of Medicine, 416A Lauder Hall 310 Cedar Street, New Haven, CT 06520, USA.
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21
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Noatynska A, Gotta M, Meraldi P. Mitotic spindle (DIS)orientation and DISease: cause or consequence? ACTA ACUST UNITED AC 2013; 199:1025-35. [PMID: 23266953 PMCID: PMC3529530 DOI: 10.1083/jcb.201209015] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Correct alignment of the mitotic spindle during cell division is crucial for cell fate determination, tissue organization, and development. Mutations causing brain diseases and cancer in humans and mice have been associated with spindle orientation defects. These defects are thought to lead to an imbalance between symmetric and asymmetric divisions, causing reduced or excessive cell proliferation. However, most of these disease-linked genes encode proteins that carry out multiple cellular functions. Here, we discuss whether spindle orientation defects are the direct cause for these diseases, or just a correlative side effect.
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Affiliation(s)
- Anna Noatynska
- Department of Physiology and Metabolism, University of Geneva, 1211 Geneva, Switzerland
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22
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Casagrande S, Ruf M, Rechsteiner M, Morra L, Brun-Schmid S, von Teichman A, Krek W, Schraml P, Moch H. The protein tyrosine phosphatase receptor type J is regulated by the pVHL-HIF axis in clear cell renal cell carcinoma. J Pathol 2013; 229:525-34. [PMID: 23007793 DOI: 10.1002/path.4107] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2012] [Revised: 08/20/2012] [Accepted: 09/12/2012] [Indexed: 11/07/2022]
Abstract
Mass spectrometry analysis of renal cancer cell lines recently suggested that the protein-tyrosine phosphatase receptor type J (PTPRJ), an important regulator of tyrosine kinase receptors, is tightly linked to the von Hippel-Lindau protein (pVHL). Therefore, we aimed to characterize the biological relevance of PTPRJ for clear cell renal cell carcinoma (ccRCC). In pVHL-negative ccRCC cell lines, both RNA and protein expression levels of PTPRJ were lower than those in the corresponding pVHL reconstituted cells. Quantitative RT-PCR and western blot analysis of ccRCC with known VHL mutation status and normal matched tissues as well as RNA in situ hybridization on a tissue microarray (TMA) confirmed a decrease of PTPRJ expression in more than 80% of ccRCCs, but in only 12% of papillary RCCs. ccRCC patients with no or reduced PTPRJ mRNA expression had a less favourable outcome than those with a normal expression status (p = 0.05). Sequence analysis of 32 PTPRJ mRNA-negative ccRCC samples showed five known polymorphisms but no mutations, implying other mechanisms leading to PTPRJ's down-regulation. Selective silencing of HIF-α by siRNA and reporter gene assays demonstrated that pVHL inactivation reduces PTPRJ expression through a HIF-dependent mechanism, which is mainly driven by HIF-2α stabilization. Our results suggest PTPRJ as a member of a pVHL-controlled pathway whose suppression by HIF is critical for ccRCC development.
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MESH Headings
- Basic Helix-Loop-Helix Transcription Factors/genetics
- Basic Helix-Loop-Helix Transcription Factors/metabolism
- Carcinoma, Renal Cell/genetics
- Carcinoma, Renal Cell/metabolism
- Carcinoma, Renal Cell/pathology
- Cell Line, Tumor
- Down-Regulation
- Gene Expression Regulation, Neoplastic
- Humans
- Hypoxia-Inducible Factor 1, alpha Subunit/genetics
- Hypoxia-Inducible Factor 1, alpha Subunit/metabolism
- In Situ Hybridization
- Kaplan-Meier Estimate
- Kidney Neoplasms/genetics
- Kidney Neoplasms/metabolism
- Kidney Neoplasms/pathology
- Models, Molecular
- Polymorphism, Genetic
- RNA, Messenger/metabolism
- RNA, Neoplasm/metabolism
- Receptor-Like Protein Tyrosine Phosphatases, Class 3/genetics
- Receptor-Like Protein Tyrosine Phosphatases, Class 3/metabolism
- Sequence Analysis, DNA
- Tissue Array Analysis
- Von Hippel-Lindau Tumor Suppressor Protein/genetics
- Von Hippel-Lindau Tumor Suppressor Protein/metabolism
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Affiliation(s)
- Silvia Casagrande
- Institute of Surgical Pathology, University Hospital Zurich, Schmelzbergstrasse 12, 8091, Zurich, Switzerland
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23
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Bader HL, Hsu T. Systemic VHL gene functions and the VHL disease. FEBS Lett 2012; 586:1562-9. [PMID: 22673568 DOI: 10.1016/j.febslet.2012.04.032] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2012] [Revised: 04/16/2012] [Accepted: 04/17/2012] [Indexed: 12/19/2022]
Abstract
The von Hippel-Lindau tumor suppressor gene (VHL) is best known as an E3 ubiquitin ligase that negatively regulates the hypoxia inducible factor (HIF). VHL mutations are the genetic defects underlying several human diseases including polycythemia, familial VHL tumor syndrome and sporadic renal cell carcinoma. VHL mutations can lead to cell-autonomous phenotypes in the tumor cells. However, non-tumor cell-autonomous functions of VHL have also been noted. VHL tumor-derived cytokines can promote inflammation and induce mobilization of endothelial progenitor cells. Up-regulation of HIF caused by VHL loss-of-function mutants, including heterozygotes, has been shown to increase the activities of hematopoietic stem cells, endothelial cells and myeloid cells. As such, systemic functions of VHL likely play important roles in the development of VHL disease.
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Affiliation(s)
- Hannah L Bader
- Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA
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24
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Teng M, Jiang XP, Zhang Q, Zhang JP, Zhang DX, Liang GP, Huang YS. Microtubular stability affects pVHL-mediated regulation of HIF-1alpha via the p38/MAPK pathway in hypoxic cardiomyocytes. PLoS One 2012; 7:e35017. [PMID: 22506063 PMCID: PMC3323643 DOI: 10.1371/journal.pone.0035017] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2011] [Accepted: 03/08/2012] [Indexed: 12/04/2022] Open
Abstract
Background Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. Methodology/Principal Findings In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule–depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. Conclusions/Significance This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.
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Affiliation(s)
| | | | | | | | | | | | - Yue-sheng Huang
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, The Third Military Medical University, Chongqing, China
- * E-mail:
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25
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Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability. Proc Natl Acad Sci U S A 2012; 109:5729-34. [PMID: 22451918 DOI: 10.1073/pnas.1108537109] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting β-tubulin, suggesting Mgr function is required for tubulin stability. Instability of β-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic β-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.
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Arjumand W, Sultana S. Role of VHL gene mutation in human renal cell carcinoma. Tumour Biol 2011; 33:9-16. [PMID: 22125026 DOI: 10.1007/s13277-011-0257-3] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2011] [Accepted: 10/25/2011] [Indexed: 12/17/2022] Open
Abstract
The Von Hippel-Lindau (VHL) is an inherited neoplasia syndrome caused by the inactivation of VHL tumor suppressor gene, and somatic mutation of this gene has been related to the development of sporadic clear cell renal carcinoma. The affected individuals are at higher risk for the development of tumor in other organs, which include pheochromocytomas, retinal angioma, pancreatic cysts, and CNS hemangioblastomas. The VHL mRNA encodes a protein (pVHL) that contains 213 amino acid residues which migrate with an apparent molecular weight of 24 to 30 kDa. The VHL gene protein has multiple functions that are linked to tumor suppression, but the best recognized and evidently linked to the development of renal cell carcinoma (RCC) is inhibition of hypoxia-inducible factor (HIF), as well as plays a role in targeting HIF for ubiquitin-mediated degradation. Aberrations in VHL's function, either through mutation or promoter hypermethylation, lead to the accumulation of HIF, which will transcriptionally upregulate a sequence of hypoxia responsive genes, including epidermal growth factor, vascular endothelial growth factor, platelet-derived growth factor, and other proangiogenic factors, resulting in upregulated blood vessel growth, one of the prerequisites of a tumor. HIF plays a critical role in pVHL-defective tumor formation, raising the possibility that drugs directed against HIF or its downstream targets (such as vascular endothelial growth factor) may one day play a role in the treatment of RCC. Moreover, a number of drugs have been developed that target HIF-responsive gene products, many of these targeted therapies have demonstrated significant activity in kidney cancer clinical trials and signify substantive advances in the treatment of this disease.
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Affiliation(s)
- Wani Arjumand
- Section of Molecular Carcinogenesis and Chemoprevention, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard, Hamdard University, Hamdard Nagar, New Delhi 110062, India.
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Complex cellular functions of the von Hippel-Lindau tumor suppressor gene: insights from model organisms. Oncogene 2011; 31:2247-57. [PMID: 21996733 DOI: 10.1038/onc.2011.442] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The von Hippel-Lindau tumor suppressor gene (VHL) has attracted intensive interest not only because its mutations predispose carriers to devastating tumors, but also because it is involved in oxygen sensing under physiological conditions. VHL loss-of-function mutations result in organ-specific tumors, such as hemangioblastoma of the central nervous system and renal cell carcinoma, both untreatable with conventional chemotherapies. The VHL protein is best known as an E3 ubiquitin ligase that targets hypoxia-inducible factor-α (HIF-α), but many diverse, non-canonical cellular functions have also been assigned to VHL, mainly based on studies in cell culture systems. As such, although the HIF-dependent role of VHL is critical, the full spectrum of pathophysiological functions of VHL is still unresolved. Such understanding requires careful cross-referencing with physiologically relevant experimental models. Studies in model systems, such as Caenorhabditis elegans, Drosophila, zebrafish and mouse have provided critical in vivo confirmation of the VHL-HIF pathway, and verification of potentially important cellular functions including microtubule stabilization and epithelial morphogenesis. More recently, animal models have also suggested systemic roles of VHL in hematopoiesis, metabolic homeostasis and inflammation. In this review, the studies performed in model organisms will be summarized and placed in context with existing clinical and in vitro data.
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Raina S, Honer M, Krämer SD, Liu Y, Wang X, Segerer S, Wüthrich RP, Serra AL. Anti-VEGF antibody treatment accelerates polycystic kidney disease. Am J Physiol Renal Physiol 2011; 301:F773-83. [PMID: 21677148 DOI: 10.1152/ajprenal.00058.2011] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Polycystic kidney growth implies expansion of the vasculature, suggesting that vascular endothelial growth factor (VEGF)-dependent processes play a critical role and that VEGF is a putative therapeutic target. Whether an anti-VEGF antibody improves renal cystic disease has not been determined. We administrated 5 mg/kg B20.4.1, an anti-VEGF-A antibody, or vehicle intraperitoneally twice weekly to 4-wk-old male normal (+/+) and cystic (Cy/+) Han:SPRD rats for 6 wk. Renal function, urinary protein excretion, organ/body weight ratios, cyst volume, tubular epithelial cell (TEC) proliferation, renal VEGF, hypoxia-inducible factor (HIF)-1α and -2α expression, renal histology, and kidney hypoxia visualized by [(18)F]fluoromisonidazole positron emission tomography were assessed. The treated compared with untreated +/+ rats had lower TEC proliferation rates, whereas Cy/+ rats receiving B20.4.1 displayed an increased proximal TEC proliferation rate, causing enhanced cyst and kidney growth. The +/+ and Cy/+ rats receiving B20.4.1 had severe renal failure and extensive glomerular damage. Proteinuria, which was highest in anti-VEGF-treated Cy/+ and lowest in untreated normal littermates, was positively correlated with renal HIF-1α and negatively correlated with VEGF expression. The untreated Cy/+ vs. +/+ rats had higher overall [(18)F]fluoromisonidazole uptake. The +/+ rats receiving B20.4.1 vs. untreated had increased [(18)F]fluoromisonidazole uptake, whereas the uptake was unchanged among treated vs. untreated Cy/+ animals. In conclusion, B20.4.1 caused an exaggerated cystic response of the proximal tubules in cystic rats and severe kidney injury that was associated with low renal VEGF and high HIF-1α levels. Anti-VEGF drug therapy may therefore not be a treatment option for polycystic kidney disease.
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Affiliation(s)
- Shagun Raina
- Zürich Center for Integrated Human Physiology, ETH Zürich, Zürich, Switzerland
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Zhou Q, Pardo A, Königshoff M, Eickelberg O, Budinger GRS, Thavarajah K, Gottardi CJ, Jones J, Varga J, Selman M, Sznajder JI, Raj JU, Zhou G. Role of von Hippel-Lindau protein in fibroblast proliferation and fibrosis. FASEB J 2011; 25:3032-44. [PMID: 21642472 DOI: 10.1096/fj.10-177824] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Idiopathic pulmonary fibrosis (IPF) is characterized by exaggerated fibroblast proliferation and accumulation of collagens and fibronectin. The extracellular fibronectin and collagen network is regulated by von Hippel-Lindau protein (pVHL). However, it is unknown whether pVHL contributes to pulmonary fibrosis. We found that lungs from patients with IPF expressed increased levels of pVHL in fibroblastic foci. Bleomycin treatment also induced pVHL in lung fibroblasts, but not in alveolar type II cells. Overexpression of pVHL increased lung fibroblast proliferation, protein abundance of fibronectin and collagen, and extracellular fibronectin. In addition, overexpression of pVHL induced expression of the α5 integrin subunit. Overexpression of pVHL did not alter hypoxia-inducible factor luciferase reporter activity and mRNA expression of vascular endothelial growth factor. Fibroblasts overexpressing pVHL were more sensitive to RGD peptide-mediated reduction in proliferation. Activating α5 and β1 integrin increased proliferation of fibroblasts overexpressing pVHL and those cells were more resistant to the inhibition of α5 integrin. Overexpression of pVHL also increased activation of focal adhesion kinase (FAK). Moreover, suppression of pVHL prevented TGF-β1-induced proliferation of mouse embryonic fibroblasts. Taken together, our results indicate that elevated expression of pVHL results in the aberrant fibronectin expression, activation of integrin/FAK signaling, fibroblast proliferation, and fibrosis.
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Affiliation(s)
- Qiyuan Zhou
- Department of Pediatrics, University of Illinois at Chicago, Chicago, IL 60612, USA
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Formenti F, Beer PA, Croft QPP, Dorrington KL, Gale DP, Lappin TRJ, Lucas GS, Maher ER, Maxwell PH, McMullin MF, O'Connor DF, Percy MJ, Pugh CW, Ratcliffe PJ, Smith TG, Talbot NP, Robbins PA. Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2alpha gain-of-function mutation. FASEB J 2011; 25:2001-11. [PMID: 21389259 PMCID: PMC3159892 DOI: 10.1096/fj.10-177378] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The hypoxia-inducible factors (HIFs; isoforms HIF-1α, HIF-2α, HIF-3α) mediate many responses to hypoxia. Their regulation is principally by oxygen-dependent degradation, which is initiated by hydroxylation of specific proline residues followed by binding of von Hippel-Lindau (VHL) protein. Chuvash polycythemia is a disorder with elevated HIF. It arises through germline homozygosity for hypomorphic VHL alleles and has a phenotype of hematological, cardiopulmonary, and metabolic abnormalities. This study explores the phenotype of two other HIF pathway diseases: classic VHL disease and HIF-2α gain-of-function mutation. No cardiopulmonary abnormalities were detected in classic VHL disease. HIF-2α gain-of-function mutations were associated with pulmonary hypertension, increased cardiac output, increased heart rate, and increased pulmonary ventilation relative to metabolism. Comparison of the HIF-2α gain-of-function responses with data from studies of Chuvash polycythemia suggested that other aspects of the Chuvash phenotype were diminished or absent. In classic VHL disease, patients are germline heterozygous for mutations in VHL, and the present results suggest that a single wild-type allele for VHL is sufficient to maintain normal cardiopulmonary function. The HIF-2α gain-of-function phenotype may be more limited than the Chuvash phenotype either because HIF-1α is not elevated in the former condition, or because other HIF-independent functions of VHL are perturbed in Chuvash polycythemia.—Formenti, F., Beer, P. A., Croft, Q. P. P., Dorrington, K. L., Gale, D. P., Lappin, T. R. J., Lucas, G. S., Maher, E. R., Maxwell, P. H., McMullin, M. F., O'Connor, D. F., Percy, M. J., Pugh, C. W., Ratcliffe, P. J., Smith, T. G., Talbot, N. P., Robbins, P. A. Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2α gain-of-function mutation.
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Affiliation(s)
- Federico Formenti
- Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Rd., Oxford, OX1 3PT, UK
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31
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Astuti D, Ricketts CJ, Chowdhury R, McDonough MA, Gentle D, Kirby G, Schlisio S, Kenchappa RS, Carter BD, Kaelin WG, Ratcliffe PJ, Schofield CJ, Latif F, Maher ER. Mutation analysis of HIF prolyl hydroxylases (PHD/EGLN) in individuals with features of phaeochromocytoma and renal cell carcinoma susceptibility. Endocr Relat Cancer 2011; 18:73-83. [PMID: 20959442 PMCID: PMC3006001 DOI: 10.1677/erc-10-0113] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/05/2022]
Abstract
Germline mutations in the von Hippel-Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-α subunits (and hence expression of the HIF-α transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (n=82) and inherited RCC (n=64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC.
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Affiliation(s)
- Dewi Astuti
- Centre for Rare Diseases and Personalised MedicineUniversity of BirminghamBirmingham, B15 2TTUK
- CRUK Renal Molecular Oncology Group, Medical and Molecular GeneticsSchool of Clinical and Experimental Medicine, Institute of Biomedical Research, University of Birmingham College of Medical and Dental SciencesBirmingham, B15 2TTUK
| | - Christopher J Ricketts
- Centre for Rare Diseases and Personalised MedicineUniversity of BirminghamBirmingham, B15 2TTUK
- CRUK Renal Molecular Oncology Group, Medical and Molecular GeneticsSchool of Clinical and Experimental Medicine, Institute of Biomedical Research, University of Birmingham College of Medical and Dental SciencesBirmingham, B15 2TTUK
| | - Rasheduzzaman Chowdhury
- Chemistry Research Laboratory, Department of ChemistryUniversity of OxfordMansfield Road, Oxford, OX1 3TAUK
| | - Michael A McDonough
- Chemistry Research Laboratory, Department of ChemistryUniversity of OxfordMansfield Road, Oxford, OX1 3TAUK
| | - Dean Gentle
- Centre for Rare Diseases and Personalised MedicineUniversity of BirminghamBirmingham, B15 2TTUK
- CRUK Renal Molecular Oncology Group, Medical and Molecular GeneticsSchool of Clinical and Experimental Medicine, Institute of Biomedical Research, University of Birmingham College of Medical and Dental SciencesBirmingham, B15 2TTUK
| | - Gail Kirby
- Centre for Rare Diseases and Personalised MedicineUniversity of BirminghamBirmingham, B15 2TTUK
- CRUK Renal Molecular Oncology Group, Medical and Molecular GeneticsSchool of Clinical and Experimental Medicine, Institute of Biomedical Research, University of Birmingham College of Medical and Dental SciencesBirmingham, B15 2TTUK
| | - Susanne Schlisio
- Howard Hughes Medical InstituteDana-Farber Cancer Institute and Brigham and Women's Hospital44 Binney Street, Boston, Massachusetts, 02115USA
- Oxygen Sensing and Cancer LaboratoryLudwig Institute for Cancer Research Ltd, Karolinska InstituteNobelsvag 3, SE-171 77, StockholmSweden
| | - Rajappa S Kenchappa
- Department of Biochemistry and Center for Molecular NeuroscienceVanderbilt University Medical SchoolNashville, Tennessee, 37232USA
| | - Bruce D Carter
- Department of Biochemistry and Center for Molecular NeuroscienceVanderbilt University Medical SchoolNashville, Tennessee, 37232USA
| | - William G Kaelin
- Howard Hughes Medical InstituteDana-Farber Cancer Institute and Brigham and Women's Hospital44 Binney Street, Boston, Massachusetts, 02115USA
| | - Peter J Ratcliffe
- Henry Wellcome Building for Molecular PhysiologyUniversity of OxfordOxford, OX3 9DUUK
| | - Christopher J Schofield
- Chemistry Research Laboratory, Department of ChemistryUniversity of OxfordMansfield Road, Oxford, OX1 3TAUK
| | - Farida Latif
- Centre for Rare Diseases and Personalised MedicineUniversity of BirminghamBirmingham, B15 2TTUK
- CRUK Renal Molecular Oncology Group, Medical and Molecular GeneticsSchool of Clinical and Experimental Medicine, Institute of Biomedical Research, University of Birmingham College of Medical and Dental SciencesBirmingham, B15 2TTUK
| | - Eamonn R Maher
- Centre for Rare Diseases and Personalised MedicineUniversity of BirminghamBirmingham, B15 2TTUK
- CRUK Renal Molecular Oncology Group, Medical and Molecular GeneticsSchool of Clinical and Experimental Medicine, Institute of Biomedical Research, University of Birmingham College of Medical and Dental SciencesBirmingham, B15 2TTUK
- West Midlands Regional Genetics ServiceBirmingham Women's Hospital, EdgbastonBirmingham, B15 2TGUK
- (Correspondence should be addressed to E R Maher, Centre for Rare Diseases and Personalised Medicine, Institute of Biomedical Research, University of Birmingham School of Medicine, Birmingham B15 2TT, UK; )
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van Rooijen E, Santhakumar K, Logister I, Voest E, Schulte-Merker S, Giles R, van Eeden F. A Zebrafish Model for VHL and Hypoxia Signaling. Methods Cell Biol 2011; 105:163-90. [DOI: 10.1016/b978-0-12-381320-6.00007-2] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Hsouna A, Nallamothu G, Kose N, Guinea M, Dammai V, Hsu T. Drosophila von Hippel-Lindau tumor suppressor gene function in epithelial tubule morphogenesis. Mol Cell Biol 2010; 30:3779-94. [PMID: 20516215 PMCID: PMC2916397 DOI: 10.1128/mcb.01578-09] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2009] [Revised: 01/22/2010] [Accepted: 05/18/2010] [Indexed: 01/16/2023] Open
Abstract
Mutations in the human von Hippel-Lindau (VHL) gene are the cause of VHL disease that displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF-alpha). However, many HIF-independent functions of VHL have been identified. Recent evidence also indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes, although it is still unclear how many of these noncanonical functions relate to the pathophysiological processes because of a lack of tractable genetic systems. Here, we report the first genomic mutant phenotype of Drosophila melanogaster VHL (dVHL) in the epithelial tubule network, the trachea, and show that dVHL regulates branch migration and lumen formation via its endocytic function. The endocytic function regulates the surface level of the chemotactic signaling receptor Breathless and promotes clearing of the lumen matrix during maturation of the tracheal tubes. Importantly, the regulatory function in tubular morphogenesis is conserved in the mammalian system, as conditional knockout of Vhl in mouse kidney also resulted in similar cell motility and lumen phenotypes.
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Affiliation(s)
- Anita Hsouna
- Department of Pathology and Laboratory Medicine and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Gouthami Nallamothu
- Department of Pathology and Laboratory Medicine and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Nurgun Kose
- Department of Pathology and Laboratory Medicine and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Maria Guinea
- Department of Pathology and Laboratory Medicine and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Vincent Dammai
- Department of Pathology and Laboratory Medicine and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Tien Hsu
- Department of Pathology and Laboratory Medicine and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
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VHL-gene deletion in single renal tubular epithelial cells and renal tubular cysts: further evidence for a cyst-dependent progression pathway of clear cell renal carcinoma in von Hippel-Lindau disease. Am J Surg Pathol 2010; 34:806-15. [PMID: 20431476 DOI: 10.1097/pas.0b013e3181ddf54d] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Inheritance of a mutant allele of the von Hippel-Lindau tumor suppressor gene predisposes affected individuals to develop renal cysts and clear cell renal cell carcinoma. Von Hippel-Lindau gene inactivation in single renal tubular cells has indirectly been showed by immunohistochemical staining for the hypoxia-inducible factor alpha target gene product carbonic anhydrase IX. In this study we were able to show von Hippel-Lindau gene deletion in carbonic anhydrase IX positive nonneoplastic renal tubular cells, in epithelial cells lining renal cysts and in a clear cell renal cell carcinoma of a von Hippel-Lindau patient. This was carried out by means of laser confocal microscopy and immunohistochemistry in combination with fluorescence in situ hybridization. Carbonic anhydrase IX negative normal renal tubular cells carried no von Hippel-Lindau gene deletion. Furthermore, recent studies have indicated that the von Hippel-Lindau gene product is necessary for the maintenance of primary cilia stability in renal epithelial cells and that disruption of the cilia structure by von Hippel-Lindau gene inactivation induces renal cyst formation. In our study, we show a significant shortening of primary cilia in epithelial cells lining renal cysts, whereas, single tubular cells with a von Hippel-Lindau gene deletion display to a far lesser extent signs of cilia shortening. Our in vivo results support a model in which renal cysts represent precursor lesions for clear cell renal cell carcinoma and arise from single renal tubular epithelial cells owing to von Hippel-Lindau gene deletion.
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Seagroves TN, Peacock DL, Liao D, Schwab LP, Krueger R, Handorf CR, Haase VH, Johnson RS. VHL deletion impairs mammary alveologenesis but is not sufficient for mammary tumorigenesis. THE AMERICAN JOURNAL OF PATHOLOGY 2010; 176:2269-82. [PMID: 20382704 PMCID: PMC2861092 DOI: 10.2353/ajpath.2010.090310] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Accepted: 01/19/2010] [Indexed: 12/21/2022]
Abstract
Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.
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Affiliation(s)
- Tiffany N Seagroves
- Department of Pathology and Laboratory Medicine, Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
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Abstract
Germ line mutations in the VHL tumor-suppressor gene cause von Hippel-Lindau (VHL) disease, a hereditary neoplastic disease associated with clear-cell renal-cell carcinomas (ccRCCs), central nervous system hemangioblastomas and pheochromocytomas. Disruption of VHL, by somatic mutation, hypermethylation of its promoter or chromosomal loss, is also seen in the majority of cases of sporadic ccRCC. The protein product of VHL, pVHL, has multiple functions, the best-documented of which relates to its ability to target hypoxia-inducible factors (HIFs) for polyubiquitination and proteasomal degradation through its role in substrate recognition as part of a ubiquitin ligase complex. Consequently, pVHL-defective ccRCCs overexpress mRNAs that are under the transcriptional control of HIF. Drugs that modulate the downstream targets of the pVHL/HIF pathway, including sunitinib, sorafenib, temsirolimus and bevacizumab, have proven benefit in treating ccRCC. In VHL disease, clear evidence supports strong genotype-phenotype correlations, but the situation in sporadic ccRCC is less clear. Data indicate that VHL alterations have a potential role as prognostic and predictive markers in ccRCC. Future clinical trials should prospectively define the VHL alteration status of study participants so that the true utility of such markers can be determined.
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Affiliation(s)
- Lucy Gossage
- Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge CB2 0RE, UK.
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37
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Duchi S, Fagnocchi L, Cavaliere V, Hsouna A, Gargiulo G, Hsu T. Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability. Development 2010; 137:1493-503. [PMID: 20388653 PMCID: PMC2853850 DOI: 10.1242/dev.042804] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/23/2010] [Indexed: 12/17/2022]
Abstract
Mutations in the human von Hippel-Lindau (VHL) genes are the cause of VHL disease, which displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF). However, many other HIF-independent functions of VHL have been identified and recent evidence indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes. Many of these functions have not been verified in genetically tractable systems. Using an established follicular epithelial model in Drosophila, we show that the Drosophila VHL gene is involved in epithelial morphogenesis via stabilizing microtubule bundles and aPKC. Microtubule defects in VHL mutants lead to mislocalization of aPKC and subsequent loss of epithelial integrity. Destabilizing microtubules in ex vivo culture of wild-type egg chambers can also result in aPKC mislocalization and epithelial defects. Importantly, paclitaxel-induced stabilization of microtubules can rescue the aPKC localization phenotype in Drosophila VHL mutant follicle cells. The results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity.
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Affiliation(s)
- Serena Duchi
- Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Selmi 3, 40126 Bologna, Italy
| | - Luca Fagnocchi
- Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Selmi 3, 40126 Bologna, Italy
| | - Valeria Cavaliere
- Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Selmi 3, 40126 Bologna, Italy
| | - Anita Hsouna
- Department of Pathology and Laboratory Medicine, and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA
| | - Giuseppe Gargiulo
- Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Selmi 3, 40126 Bologna, Italy
| | - Tien Hsu
- Department of Pathology and Laboratory Medicine, and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA
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Chacon‐Camacho OF, Rodriguez‐Dennen F, Camacho‐Molina A, Rasmussen A, Alonso‐Vilatela E, Zenteno JC. Clinical and molecular features of familial and sporadic cases of von Hippel‐Lindau disease from Mexico. Clin Exp Ophthalmol 2010; 38:277-83. [DOI: 10.1111/j.1442-9071.2010.02241.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Affiliation(s)
| | | | | | - Astrid Rasmussen
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA,
| | - Elisa Alonso‐Vilatela
- Department of Neurogenetics and Molecular Biology, Instituto Nacional de Neurología y Neurocirugía ‘Manuel Velasco Suarez’, Mexico City, Mexico, and
| | - Juan C Zenteno
- Department of Genetics,
- Department of Biochemistry, Faculty of Medicine, National University Autonomous of Mexico, Mexico City, Mexico
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Abstract
Geneticists estimate that 5% to 10% of all cancers diagnosed in the pediatric age range occur in children born with a genetic mutation that directly increases their lifetime risk for neoplasia. However, despite the fact that only a fraction of cancers in children occur as a result of an identified inherited predisposition, characterizing genetic mutations responsible for increased cancer risk in such syndromes has resulted in a profound understanding of relevant molecular pathways involved in carcinogenesis and/or resistance to neoplasia. Importantly, because most cancer predisposition syndromes result in an increased risk of a small number of defined malignancies, personalized prophylactic surveillance and preventive measures can be implemented in affected patients. Lastly, many of the same genetic targets identified from cancer-prone families are mechanistically involved in the majority of sporadic cancers in adults and children, thereby underscoring the clinical relevance of knowledge gained from these defined syndromes and introducing novel therapeutic opportunities to the broader oncologic community. This review highlights the clinical and genetic features of many of the known constitutional genetic syndromes that predispose to malignancy in children and young adults.
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40
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Ferchichi I, Stambouli N, Marrackchi R, Arlot Y, Prigent C, Fadiel A, Odunsi K, Ben Ammar Elgaaied A, Hamza A. Experimental and computational studies indicate specific binding of pVHL protein to Aurora-A kinase. J Phys Chem B 2010; 114:1486-97. [PMID: 20047310 DOI: 10.1021/jp909869g] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Overexpression of Aurora-A kinase is commonly detected in many cancers, whereas the von Hippel-Lindau protein (pVHL) is frequently mutated or absent in renal cell carcinoma and is involved in the Ub proteasome complex, an important degradation pathway. In order to establish a link between Aurora-A overexpression and lack of pVHL protein, we hypothesized that pVHL regulates Aurora-A expression through a physical interaction. We present the first evidence, from both biological assays and computational biology techniques, that human pVHL binds strongly to Aurora-A kinase. Extensive molecular modeling, docking, and dynamic simulations demonstrate that the structure of the pVHL protein would allow it to bind to the TPX2 binding region of Aurora-A. In view of Aurora-A's importance as a therapeutic target for the treatment of cancer, this observation provides novel insights into the Aurora-A/pVHL pathway. In addition, the detailed Aurora-A/pVHL binding structure obtained will be valuable for the design of future Aurora-A inhibitors as therapeutic agents.
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Affiliation(s)
- Imen Ferchichi
- Laboratory of Genetics, Immunology and Human Pathology, Faculty of Sciences of Tunis, Tunisia
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41
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Dose-dependent modulation of HIF-1alpha/sima controls the rate of cell migration and invasion in Drosophila ovary border cells. Oncogene 2009; 29:1123-34. [PMID: 19966858 DOI: 10.1038/onc.2009.407] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The role of the hypoxic response during metastasis was analysed in migrating border cells of the Drosophila ovary. Acute exposure to 1% O(2) delayed or blocked border cell migration (BCM), whereas prolonged exposure resulted in the first documented accelerated BCM phenotype. Similarly, manipulating the expression levels of sima, the Drosophila hypoxia-inducible factor (HIF)-1alpha ortholog, revealed that Sima can either block or restore BCM in a dose-dependent manner. In contrast, over-expression of Vhl (Drosophila von Hippel-Lindau) generated a range of phenotypes, including blocked, delayed and accelerated BCM, whereas over-expression of hph (Drosophila HIF prolyl hydroxylase) only accelerated BCM. Mosaic clone analysis of sima or tango (HIF-1beta ortholog) mutants revealed that cells lacking Hif-1 transcriptional activity were preferentially detected in the leading cell position of the cluster, resulting in either a delay or acceleration of BCM. Moreover, in sima mutant cell clones, there was reduced expression of nuclear slow border cells (Slbo) and basolateral DE-cadherin, proteins essential for proper BCM. These results show that Sima levels define the rate of BCM in part through regulation of Slbo and DE-cadherin, and suggest that dynamic regulation of Hif-1 activity is necessary to maintain invasive potential of migrating epithelial cells.
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Forman JR, Worth CL, Bickerton GRJ, Eisen TG, Blundell TL. Structural bioinformatics mutation analysis reveals genotype-phenotype correlations in von Hippel-Lindau disease and suggests molecular mechanisms of tumorigenesis. Proteins 2009; 77:84-96. [PMID: 19408298 DOI: 10.1002/prot.22419] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Mutations in the VHL gene lead to von Hippel-Lindau (VHL) disease, a clinically heterogeneous cancer syndrome. Here, we use software and database tools to understand and predict the phenotypes associated with missense mutations in the VHL gene product, pVHL. The protein product pVHL is known to interact with elongin B, elongin C, and the HIF substrate. By analyzing known and predicted interaction sites and predictions of thermodynamic stability change upon mutation, we generate new hypotheses regarding the molecular etiology of renal cell carcinoma (RCC) and pheochromocytoma (PCC) in VHL disease. We find that the molecular causes of RCC and PCC appear to be decoupled. RCC may arise through two distinct mechanisms: disruption of HIF interactions or binding at the elongin B interface. PCC is triggered by mutations which disrupt interactions at the elongin C binding site. These findings have important implications for VHL disease and for nonfamilial RCC, because most cases of clear cell RCC are linked with VHL inactivation. Additionally, predicting effects of genetic variation will be critical as genetic sequencing accelerates; the analytical strategy presented here may elucidate other systems as further data on genetic variation become available.
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Affiliation(s)
- Julia R Forman
- Biocomputing Group, Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
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43
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Adding structural information to the von Hippel-Lindau (VHL) tumor suppressor interaction network. FEBS Lett 2009; 583:3704-10. [PMID: 19878677 DOI: 10.1016/j.febslet.2009.10.070] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2009] [Revised: 10/21/2009] [Accepted: 10/26/2009] [Indexed: 11/23/2022]
Abstract
The von Hippel-Lindau (VHL) tumor suppressor gene is a protein interaction hub, controlling numerous genes implicated in tumor progression. Here we focus on structural aspects of protein interactions for a list of 35 experimentally verified protein VHL (pVHL) interactors. Using structural information and computational analysis we have located three distinct interaction interfaces (A, B, and C). Interface B is the most versatile, recognizing a refined linear motif present in 17 otherwise non-related proteins. It has been possible to distinguish compatible and exclusive interactions by relating pVHL function to interaction interfaces and subcellular localization. A novel hypothesis is presented regarding the possible function of the N-terminus as an inhibitor of pVHL function.
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44
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Nallamothu G, Dammai V, Hsu T. Developmental function of Nm23/awd: a mediator of endocytosis. Mol Cell Biochem 2009; 329:35-44. [PMID: 19373545 PMCID: PMC2721904 DOI: 10.1007/s11010-009-0112-7] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2009] [Accepted: 04/02/2009] [Indexed: 10/20/2022]
Abstract
The metastasis suppressor gene Nm23 is highly conserved from yeast to human, implicating a critical developmental function. Studies in cultured mammalian cells have identified several potential functions, but many have not been directly verified in vivo. Here, we summarize the studies on the Drosophila homolog of the Nm23 gene, named a bnormal w ing d iscs (awd), which shares 78% amino acid identity with the human Nm23-H1 and H2 isoforms. These studies confirmed that awd gene encodes a nucleoside diphosphate kinase, and provided strong evidence of a role for awd in regulating cell differentiation and motility via regulation of growth factor receptor signaling. The latter function is mainly mediated by control of endocytosis. This review provides a historical account of the discovery and subsequent analyses of the awd gene. We will also discuss the possible molecular function of the Awd protein that underlies the endocytic function.
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Affiliation(s)
- Gouthami Nallamothu
- Department of Pathology and Laboratory Medicine, and Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas St., Charleston, SC 29425, USA
| | - Vincent Dammai
- Department of Pathology and Laboratory Medicine, and Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas St., Charleston, SC 29425, USA
| | - Tien Hsu
- Department of Pathology and Laboratory Medicine, and Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas St., Charleston, SC 29425, USA
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Luu VD, Boysen G, Struckmann K, Casagrande S, von Teichman A, Wild PJ, Sulser T, Schraml P, Moch H. Loss of VHL and Hypoxia Provokes PAX2 Up-Regulation in Clear Cell Renal Cell Carcinoma. Clin Cancer Res 2009; 15:3297-304. [DOI: 10.1158/1078-0432.ccr-08-2779] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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Secades P, Rodrigo JP, Hermsen M, Alvarez C, Suarez C, Chiara MD. Increase in gene dosage is a mechanism of HIF-1alpha constitutive expression in head and neck squamous cell carcinomas. Genes Chromosomes Cancer 2009; 48:441-54. [PMID: 19235921 DOI: 10.1002/gcc.20652] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
The HIF-1alpha protein plays a key role in the cellular response to hypoxia via transcriptional regulation of genes involved in erythropoiesis, angiogenesis, and metabolism. Overexpression of HIF-1alpha is commonly found in solid tumors in significant association with increased patient mortality and resistance to therapy. The predominant mode of HIF-1alpha regulation by hypoxia occurs at the level of protein stability. In addition to hypoxia, HIF-1alpha protein stability and synthesis is regulated by nonhypoxic signals such as inactivation of tumor suppressors and activation of oncogenes. Here, we show that an increase in gene dosage may contribute to HIF-1alpha mRNA and protein overexpression in a nonhypoxic environment in head and neck squamous cell carcinomas (HNSCC). Increased HIF-1alpha gene dosage was found in one out of five HNSCC-derived cell lines and three out of 27 HNSCC primary tumors. Significantly, increased gene dosage in those samples was associated with high HIF-1alpha mRNA and protein levels. Normoxic overexpression of HIF-1alpha protein in HNSCC-derived cell lines was also paralleled by higher expression levels of HIF-1alpha target genes. Array CGH analysis confirmed the copy number increase of HIF-1alpha gene and revealed that the gene is contained within a region of amplification at 14q23-q24.2 both in the cell line and primary tumors. In addition, FISH analysis revealed the presence of 11-13 copies on a tetraploid background in SCC2 cells. These data suggest that increased HIF-1alpha gene dosage is a mechanism of HIF-1alpha protein overexpression in HNSCC that possibly prepares the cells for a higher activity in an intratumoral hypoxic environment.
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Affiliation(s)
- Pablo Secades
- Servicio de Otorrinolaringología, Hospital Universitario Central de Asturias
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47
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Knauth K, Cartwright E, Freund S, Bycroft M, Buchberger A. VHL mutations linked to type 2C von Hippel-Lindau disease cause extensive structural perturbations in pVHL. J Biol Chem 2009; 284:10514-22. [PMID: 19228690 PMCID: PMC2667738 DOI: 10.1074/jbc.m809056200] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2008] [Indexed: 11/06/2022] Open
Abstract
pVHL (von Hippel-Lindau tumor suppressor protein) is the substrate recognition subunit of the CBC(VHL) ubiquitin ligase complex promoting the degradation of hypoxia-inducible factor subunits, HIF-1/2alpha. Mutational inactivation of pVHL causes the hereditary von Hippel-Lindau tumor syndrome, which predisposes affected individuals to hemangioblastomas, renal cell carcinomas, and pheochromocytomas. Whereas the development of hemangioblastomas and renal cell carcinomas has been attributed to impaired HIF-1/2alpha down-regulation by pVHL mutant proteins, the molecular defects underlying the development of pheochromocytomas are still unknown. Here, we present a detailed biochemical analysis of pVHL mutant proteins linked to type 2C (pheochromocytoma only) von Hippel-Lindau disease. Type 2C-associated mutations caused extensive structural perturbations of pVHL, as revealed by the reduced stability, increased proteolytic susceptibility, and dramatically altered NMR spectrum of recombinant, mutant pVHL-ElonginC-ElonginB complexes in vitro. In human cell lines, type 2C-linked mutations destabilized the CBC(VHL) ubiquitin ligase complex and resulted in reduced cellular pVHL levels. Together, our data reveal unexpectedly strong structural defects of type 2C-associated pVHL mutant proteins that are likely to affect both HIF-1/2alpha-related and -unrelated pVHL functions in the pathogenesis of pheochromocytomas.
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Affiliation(s)
- Katja Knauth
- Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
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48
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Lee KH, Lee JS, Kim BJ, Lee JK, Kim SH, Kim SH, Lee KT. Pancreatic involvement in Korean patients with von Hippel-Lindau disease. J Gastroenterol 2009; 44:447-452. [PMID: 19333546 DOI: 10.1007/s00535-009-0021-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/04/2008] [Accepted: 11/29/2008] [Indexed: 02/04/2023]
Abstract
PURPOSE The aim of this study was to describe pancreatic involvement in von Hippel-Lindau (VHL) disease and to document the changes that occur in pancreatic lesions. METHODS We retrospectively analyzed the medical records and CT scans of 18 VHL patients who were diagnosed between 1994 and 2007 at the Samsung Medical Center. The clinical history with a detailed family history, biochemical test results, and imaging studies of the pancreas, adrenal glands, and kidneys were reviewed. Genetic analysis was performed in 12 patients. The changes in pancreatic lesions, such as an increase in cystic lesions, calcifications, and dilatation of the pancreatic duct, were analyzed in patients who had CT scans at least 1 year apart. RESULTS Pancreatic lesions existed in 89% (16/18) of the patients. All 16 patients had multiple cystic lesions. Two patients had co-existing neuroendocrine tumors (NET), and two patients had co-existing serous cystadenomas (SCA). At least one of three features of pancreatic lesions (cystic lesions, calcifications, and dilatation of the pancreatic duct) progressed in all nine patients who had CT scans 1 year apart. CONCLUSION Pancreatic involvement in VHL disease was relatively common in Korean patients. The most common type of pancreatic involvement was a multiple cystic lesion. NET and SCA existed in approximately 10% of VHL patients with pancreatic involvement. Pancreatic lesions in VHL disease progressed, at least according to radiological images.
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Affiliation(s)
- Kwang Hyuck Lee
- Department of Internal Medicine, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Gangnam-gu, Seoul, Korea
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49
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Iguchi M, Kakinuma Y, Kurabayashi A, Sato T, Shuin T, Hong SB, Schmidt LS, Furihata M. Acute inactivation of the VHL gene contributes to protective effects of ischemic preconditioning in the mouse kidney. NEPHRON. EXPERIMENTAL NEPHROLOGY 2008; 110:e82-90. [PMID: 18957870 PMCID: PMC2657226 DOI: 10.1159/000166994] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 02/07/2008] [Accepted: 07/30/2008] [Indexed: 01/20/2023]
Abstract
BACKGROUND/AIMS The von Hippel-Lindau (VHL) protein functions as an E3 ubiquitin ligase, controlling the stability of hypoxia-inducible factor (HIF). Preinduction of HIF-1alpha before pathological insult activates a self-defense mechanism and suppresses further aggravation of organ or cellular injury by ischemia. We investigated whether acute inactivation of the VHL gene might play a role in the response of mice to ischemic renal injury. METHODS We generated tamoxifen-inducible conditional VHL knockout (VHL-KO) mice to inactivate the VHL gene in an acute manner during renal ischemia-reperfusion injury (IRI) induced by bilateral clamping of kidney arteries. Renal IRI is characterized by renal dysfunction and tubular damage. RESULTS After the procedure of IRI, blood urea nitrogen (BUN) and creatinine (CRN) levels in control mice were significantly higher (BUN, 138.10 +/- 13.03 mg/dl; CRN, 0.72 +/- 0.16 mg/dl) than in VHL-KO mice (BUN, 52.12 +/- 6.61 mg/dl; CRN, 0.24 +/- 0.04 mg/dl; BUN: p < 0.05; CRN: p < 0.05). Histologically, tubular injury scores were higher in control mice than in VHL-KO mice (p < 0.05). CONCLUSION We suggest that the acute inactivation of the VHL gene contributes to protective effects of ischemic preconditioning in renal tubules of the mouse.
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Affiliation(s)
- Mitsuko Iguchi
- Department of Pathology, Kochi Medical School, Nankoku, Kochi, Japan
| | - Yoshihiko Kakinuma
- Department of Cardiovascular Control, Kochi Medical School, Nankoku, Kochi, Japan
| | | | - Takayuki Sato
- Department of Cardiovascular Control, Kochi Medical School, Nankoku, Kochi, Japan
| | - Taro Shuin
- Department of Urology, Kochi Medical School, Nankoku, Kochi, Japan
| | - Seung-Beom Hong
- Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
| | - Laura S. Schmidt
- Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
- Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD, USA
| | - Mutsuo Furihata
- Department of Pathology, Kochi Medical School, Nankoku, Kochi, Japan
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50
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Aune GJ, Takagi K, Sordet O, Guirouilh-Barbat J, Antony S, Bohr VA, Pommier Y. Von Hippel-Lindau-coupled and transcription-coupled nucleotide excision repair-dependent degradation of RNA polymerase II in response to trabectedin. Clin Cancer Res 2008; 14:6449-55. [PMID: 18927284 PMCID: PMC2662138 DOI: 10.1158/1078-0432.ccr-08-0730] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Ecteinascidin 743 (Et743; trabectedin, Yondelis) has recently been approved in Europe for the treatment of soft tissue sarcomas and is undergoing clinical trials for other solid tumors. Et743 selectively targets cells proficient for TC-NER, which sets it apart from other DNA alkylating agents. In the present study, we examined the effects of Et743 on RNA Pol II. EXPERIMENTAL DESIGN AND RESULTS We report that Et743 induces the rapid and massive degradation of transcribing Pol II in various cancer cell lines and normal fibroblasts. Pol II degradation was abrogated by the proteasome inhibitor MG132 and was dependent on TC-NER. Cockayne syndrome (CS) cells and xeroderma pigmentosum (XP) cells (XPD, XPA, XPG, and XPF) were defective in Pol II degradation, whereas XPC cells whose defect is limited to global genome NER in nontranscribing regions were proficient for Pol II degradation. Complementation of the CSB and XPD cells restored Pol II degradation. We also show that cells defective for the VHL complex were defective in Pol II degradation and that complementation of those cells restores Pol II degradation. Moreover, VHL deficiency rendered cells resistant to Et743-induced cell death, a similar effect to that of TC-NER deficiency. CONCLUSION These results suggest that both TC-NER-induced and VHL-mediated Pol II degradation play a role in cell killing by Et743.
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Affiliation(s)
- Gregory J. Aune
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Kazutaka Takagi
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Olivier Sordet
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Jose Guirouilh-Barbat
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Smitha Antony
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Vilhelm A. Bohr
- Laboratory of Molecular Gerontology, National Institute of A ging, NIH, Baltimore, Maryland
| | - Yves Pommier
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
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